Actin assembly is intimately linked to other developmental and physiological programs, including cell division, secretion, motility and differentiation. Much of the linkage between actin function and other cellular functions occurs through intermediate messengers with broad specificity such as Cdc42, Rac, and Nck. We have investigated in detail how these proteins affect actin nucleation through N-WASP and WAVE and this has recently led to the identification of complexes with other proteins that regulate actin nucleation via Rac and Nck. We propose to study the structure and composition of these complexes in both brain extracts and in neutrophils with the aim of determining how they might coordinately regulate other cellular activities, such as protein translation. We plan to study a component of the WAVE complex, a newly discovered actin capping protein, HSPC300, which on association with WAVE may affect the morphology of actin branching patterns in cells. A second major aim of this proposal is to understand the function of a newly discovered protein essential for actin nucleation and elongation in Xenopus extracts, called MCAP2b. This protein, conserved in all metazoans, lacks a typical CRIB domain but binds to Cdc42GTP. MCAP2b is required to promote assembly in extracts but has no activity in reconstituted systems involving N-WASP, Arp2,3 and activators. The activity of this protein has exposed our general ignorance of actin dynamics in cellular systems and is the basis for considerable work in this proposal using total internal reflectance fluorescence microscopy in complete cellular extracts. Finally, we ask why actin does not polymerize in response to Rac in eggs and early embryos but acquire that capacity at gastrulation. These studies are aimed at understanding actin regulation in a cellular context and the integration of actin regulation with other cellular functions.